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Pone.0064711 1.15

Increased Beta2-Adrenoceptors in Doxorubicin-InducedCardiomyopathy in Rat
Nolwenn Merlet1,2, Nicolas Piriou1,3, Bertrand Rozec1,4, Amandine Grabherr1, Benjamin Lauzier1,2, Jean-
Noe¨l Trochu1,2,3, Chantal Gauthier1,2*
1 l'institut du thorax, Unite´ Inserm UMR 1087/CNRS UMR 6291, Nantes, France, 2 Universite´ de Nantes, Nantes, France, 3 CHU Nantes, l'institut du thorax, Nantes, France,
4 CHU Nantes, Department of Anaesthesiology, Nantes, France
Background: The toxicity of doxorubicin, leading to an irreversible heart failure, limits its use as chemotherapeutic agent.The beneficial effects of early administration of b-blocker were reported in patients with heart failure due to doxorubicin,suggesting an important role of b-adrenoceptors (b-ARs). This study aimed to identify a putative target (b-AR and/or itseffectors) at the early phase of a chronic doxorubicin-induced cardiomyopathy (Dox-CM) in a rat model.
Methodology: Dox-CM was induced by six doxorubicin injections (cumulative dose: 15 mg.kg21) and validated byechocardiography and left ventricle (LV) catheterization. The b-AR protein expressions in LV were evaluated by western-blotat days 35 (d35) and 70 (d70) after the first doxorubicin injection. Ex vivo cardiac contractility (dP/dtmax, dP/dtmin) wasevaluated on isolated heart in response to specific b-AR stimulations at d35.
Results: At d35, Dox-CM hearts were characterized by mild LV systolic and diastolic dysfunctions, which were exacerbated atd70. In Dox-CM hearts, b3-AR expression was only decreased at d70 (-3768%). At d35, b1-AR expression was decreased by6866%, but ex vivo b1-AR function was preserved due to, at least in part, an increased adenylyl cyclase response assessed byforskolin. b2-AR expression was increased both at d35 (+58622%) and d70 (+174635%), with an increase of ex vivo b2-ARresponse at d35. Inhibition of Gi protein with pertussis toxin did not affect b2-AR response in Dox-CM hearts, suggesting adecoupling of b2-AR to Gi protein.
Conclusion: This study highlights the b1/b2-AR imbalance in early Dox-CM and reveals the important role that b2-AR/Gicoupling could play in this pathology. Our results suggest that b2-AR could be an interesting target at early stage of Dox-CM.
Citation: Merlet N, Piriou N, Rozec B, Grabherr A, Lauzier B, et al. (2013) Increased Beta2-Adrenoceptors in Doxorubicin-Induced Cardiomyopathy in Rat. PLoSONE 8(5): e64711. doi:10.1371/journal.pone.0064711
Editor: Philippe Rouet, I2MC INSERM UMR U1048, France
Received January 29, 2013; Accepted April 17, 2013; Published May 31, 2013
Copyright: ß 2013 Merlet et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the ‘‘Association Franc¸aise contre les Myopathies'', the ‘‘Fe´de´ration Franc¸aise de Cardiologie'', the ‘‘Fondation de France''and the ‘‘Fondation Genavie''. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: chantal.gauthier@univ-nantes.fr
As in different HF etiologies, Dox-CM is characterized by an
alteration of adrenergic system [8]. However, at the present time,
Anthracyclines, like doxorubicin (Dox), epirubicin and dauno-
only few studies have examined the role of cardiac b1- and b2-
rubicin, are among the most effective drugs used in chemotherapy
adrenoceptor (b-AR) subtypes in the pathogenesis of Dox-
for cancer patients. Since the late 60s, Dox is frequently used
cardiotoxicity [9,10] and only one study, at late-onset Dox-CM,
against a variety of cancers including Hodgkin's lymphoma [1],
assessed b3-AR subtype [11], which is recently described as a new
soft-tissue sarcomas [2], leukemia and solid tumors. However, Dox
target for some b-blockers such as nebivolol [12,13]. Despite this
administration is limited due to severe cardiotoxic effects leading
lack in experimental data, some clinical studies investigated b-
to dilated cardiomyopathy [3]. Prognosis of heart failure (HF) due
blocker therapies in Dox-CM. Kalay et al., demonstrated that left
to Dox-cardiotoxicity is poor and even worse than ischemic or
ventricular (LV) diameters remained constant and diastolic
idiopathic dilated cardiomyopathy. Although several mechanisms
function was better preserved after Dox-treatment in patients
have been proposed to describe the mechanisms by which Dox
receiving carvedilol, compared to placebo [14]. However,
induces cardiotoxicity (generation of free radicals, mitochondrial
Georgakopoulos et al., demonstrated that metoprolol, a b-blocker
disruption, alteration of cellular energetic, and initiation of
without antioxidative properties, failed to give cardioprotection in
apoptotic cascades), these mechanisms are still not fully under-
lymphoma-treated doxorubicin patients [15]. It was reported that
stood [4–6] and there is no specific treatment for Dox-induced
an early start of treatment with angiotensin-converting enzyme
cardiomyopathy (Dox-CM) [6,7]; treatments classically used for
inhibitors (ACEIs), in association or not with b-blockers, both
other HFs with systolic dysfunction induce only limited beneficial
improves myocardial contractility [16] and patients' prognosis
effects in Dox-CM.
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May 2013 Volume 8 Issue 5 e64711
Beta-Adrenoceptors in Doxorubicin Cardiomyopathy
Table 1. Antibodies used for western-blot.
Secondary antibody
Rabbit polyclonal antibody, Sigma-Aldrich
Goat anti-rabbit immunoglobulin G, Santa-Cruz
Biotechnology (sc-2054)
Rabbit polyclonal antibody, AbCam Ltd
Goat anti-rabbit immunoglobulin G, Santa-Cruz
Biotechnology (sc-2054)
Rabbit polyclonal antibody, Santa Cruz
Goat anti-rabbit immunoglobulin G, Santa-Cruz
Biotechnology (sc-50436)
Biotechnology (sc-2054)
Rabbit polyclonal antibody, Merk Chemicals Ltd 1/1,000
Goat anti-rabbit immunoglobulin G, Santa-Cruz
Biotechnology (sc-2054)
Rabbit polyclonal antibody, Merk Chemicals Ltd 1/5,000
Goat anti-rabbit immunoglobulin G, Santa-Cruz
Biotechnology (sc-2054)
Mouse monoclonal antibody, Santa-Cruz
Goat anti-mouse immunoglobulin G, Santa-Cruz
Biotechnology (sc-32233)
Biotechnology (sc-2055)
Primary antibodies were diluted in TBS-T, excepted for Gia2 protein detection which was diluted in 5% non-fat dry milk in TBS-T. All secondary antibodies were diluted in1% non-fat dry milk in TBS-T.doi:10.1371/journal.pone.0064711.t001
[17]. Although the exact mechanism is still poorly understood, theauthors highlighted the importance of an early diagnosis, becausea delayed treatment (.6 months after the end of chemotherapy) isinefficient [17].
The aim of the present study was to identify a putative target (b-
AR and/or its effectors) involved at the early phase of a chronicDox-CM in a rat model. This study demonstrated for the first timethat b2-AR expression was increased from 35 days after the firstDox-injection, this effect was maintained until 70 days after thefirst Dox-injection, resulting in an increase of b2-AR-inducedcontractility. In addition, b1-AR function was preserved, in spite ofdecreased b1-AR protein expression. This discrepancy could beexplained by an increase of adenylyl cyclase (AC) expression and/or activity as illustrated by an increased forskolin-induced responsein Dox-CM rats.
All experiments were performed in accordance with the 1996
Guide for the Care and Use of Laboratory Animals published bythe U.S. National Institute of Health. The protocol was approvedby the Direction De´partementale de la Protection des Populations(agreement number C-44 015) and all efforts were made tominimize suffering.
One hundred and fifty eight male Sprague-Dawley rats (225–
250 g) were purchased from Janvier (Le Genest St Isle, France)and were housed under standard conditions of room temperature,humidity (40–60%) and 12 h light/dark cycle. Food and waterwere available ad libitum.
Doxorubicin (AdriblastineH 50 mg/25 mL, Pfizer, France) was
administered intraperitoneally in six equal injections (eachcontaining 2.5 mg.kg21) over a period of two weeks, with a totalcumulative dose of 15 mg.kg21 body weight (Dox-CM: n = 100).Age-matched rats injected with saline were used as controls (Ctrl:n = 58). Rats were then bred in animal housing until three weeksafter the last injection (day 35 (d35); Ctrl: n = 46, Dox-CM: n = 76)
Figure 1. Body weight (A) and survival curves (B) of Ctrl and
or until eight weeks after the last injection (day 70 (d70); Ctrl:
Dox-CM rats. Values are means 6 sem **: P,0.001 vs Ctrl. Ctrl:
n = 12, Dox-CM: n = 24). At d35, twenty Dox-CM rats and twenty
control, Dox-CM: Doxorubicin-induced cardiomyopathy.
Ctrl rats were randomly selected to be hemodynamically explored
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Beta-Adrenoceptors in Doxorubicin Cardiomyopathy
Table 2. Cardiac morphological parameters in Ctrl and Dox-CM rats, at d35 and d70.
Heart wt/Body wt (mg.g21)
LV wt/Body wt (mg.g21)
Heart wt/Tibia length (mg.cm21)
LV wt/Tibia length (mg.cm21)
Ctrl: control; Dox-CM: Doxorubicin-induced cardiomyopathy; LV: left ventricle; wt: weight. Values are means 6 sem. *: P,0.05 vs respective Ctrl. **: P,0.001 vsrespective Ctrl.doi:10.1371/journal.pone.0064711.t002
by echocardiography-Doppler. Then either rats were hemody-
2D speckle-tracking method on every medial myocardial segment
namically assessed by LV catheterization, or rat hearts were
removed either to test ex vivo cardiac contractile function or toperform biochemical studies. At d70, rats were used to perform
Left Ventricle Catheterization
echocardiography-Doppler and biochemical studies.
LV catheterization was performed, at d35, by a 2F microtip
pressure catheter (SPR 838, Millar instruments Inc, Houston,
Texas). Anaesthesia maintenance on spontaneously breathing rats
Transthoracic echocardiography was performed using a com-
was performed with an inhalational anaesthesia system for small
mercially available ultrasound system (VIVID7, GE Healthcare,
animal (TEM anaesthesia, Lormont, France). Isoflurane was
Horton, Norway) equipped with a 10 MHz sectorial probe. Rats
delivered through a nose mask at a concentration of 2% volume
were anaesthetized with a gas-mixture of 1% isoflurane (ForeneH,
and 1 L.min21 O2 flow to limit hemodynamic repercussion. Body
Abbott France, Rungis, France) in O2. The chest was shaved and
temperature was monitored by rectal probe and maintained
the animal was positioned on a heating pad in a supine position.
constant (37uC) by warming-blanket. The right carotid artery was
All recordings were monitored under a continuous single-channel
isolated, ligated at the proximal part and the pressure catheter was
electrocardiogram obtained on the imaging system by fixing the
inserted in. Signals were recorded using an Analogic/Digital
electrodes to the limbs. Using two-dimensional imaging, a short
converter (EMKA Technologies, Paris, France), stored and
axis view of the LV at the level of the papillary muscles was
displayed on a computer by the IOX1.5.7 Software System
obtained and the two-dimensionally guided M-mode recording
(EMKA Technologies). Data were analysed using Datanalyst
through the anterior and posterior walls of the LV was taken as
software (EMKA Technologies). The following parameters were
recommended by the American Society of Echocardiography [18].
obtained: LV end diastolic pressure (LVEDP), LV contraction and
Then, trans-mitral inflow in pulsed-wave Doppler from apical four
relaxation velocities (dP/dtmax and dP/dtmin, respectively), the
chamber view and tissue Doppler imaging (TDI) on basal
index of LV relaxation constant (Tau).
segments of septal and lateral walls in apical four chamber view
The animals were thereafter sacrificed by injection of a
were taken as previously described [19]. A cine-loop of LV
parasternal short axis view with high frame rate was obtained. Allacquisitions were performed by the same operator.
All images were digitally stored on hard disks for off-line analysis
The expressions of b1-AR, b2-AR, b3-AR, Gsa, Gia2 and
(EchoPac Q-analysis software, GE Healthcare). Measurements
GAPDH were examined by western-blot, at d35 and d70. Hearts
were made on five cardiac cycles and averaged for each data
were rapidly isolated and placed in a cold Tyrode solution
value. The following parameters were determined as recom-
composed as followed (in mM): NaCl, 137; KCl, 5.4; MgCl2, 1.2;
mended by the American Society of Echocardiography [18]: LV
Na2HPO4, 1.2; Hepes, 20; CaCl2, 1.0; pH 7.4 (Sigma-Aldrich, St
end diastolic and systolic diameters (LVEDD and LVESD),
Quentin Fallavier, France). LV and septum free walls were
diastolic posterior wall thicknesses (dPWth). LV end diastolic and
carefully separated and freezed in liquid nitrogen and stored at
systolic volumes (LVEDV and LVESV) were calculated from the
280uC until used. LV and septum free walls samples were
Teichholz method in order to assess LV ejection fraction (LVEF),
homogenized in 3 mL of Ripa buffer for 1 g of tissue plus 1X
whereas LV shortening fraction (LVSF) was calculated from
PMSF (Interchim, Montluc¸on, France) and 2X protease inhibitor
LVEDD and LVESD previously measured. LV diastolic function
cocktail (Roche, Mannheim, Germany). Protein samples (25 mg)
parameters were derived from pulsed-wave trans-mitral inflow
were submitted to electrophoresis on a 10% polyacrylamide/
pattern and TDI off-line analyses as previously described [20]: the
sodium dodecyl sulfate gel and then were run 150 min at 20 mA
peak of E wave velocities, the isovolumic relaxation time (IVRT),
per membrane in TG-SDS buffer (Interchim). Gels and nitrocel-
the mean of peak velocities of basal septal and lateral walls (pulsed
lulose membranes (Hybond C super membrane, Amersham, Saclay,
wave TDI) during systole (Sa) and in early diastole (Ea) to calculate
France) were equilibrated in TG-SDS buffer with 20% ethanol,
E/Ea ratio. Radial 2D strain analyses were performed using the
and protein fractions were transferred using an electroblottingapparatus (Bio-Rad, Marnes La Coquette, France). Nonspecific
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Beta-Adrenoceptors in Doxorubicin Cardiomyopathy
Figure 2. Representative images obtained by echocardiography at day 35 in Ctrl (A) and Dox-CM (B) rats. Images were obtained with ashort axis view of a two-dimensionally directed M-mode. Ctrl: control, dAWth: diastolic anterior wall thickness, Dox-CM: Doxorubicin-inducedcardiomyopathy, dPWth: diastolic posterior wall thickness, LVEDD: left ventricular end diastolic diameter, LVESD: left ventricular end systolic diameter,sAWth: systolic anterior wall thickness, sPWth: systolic posterior wall thickness.doi:10.1371/journal.pone.0064711.g002
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Beta-Adrenoceptors in Doxorubicin Cardiomyopathy
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Beta-Adrenoceptors in Doxorubicin Cardiomyopathy
Figure 3. In vivo cardiac parameters obtained by echocardiography-Doppler in Ctrl and Dox-CM rats. Ctrl are showed in empty bar andDox-CM in full bar. Values are means 6 sem *: P,0.05 vs respective Ctrl **: P,0.001 vs respective Ctrl. d35: day 35, d70: day 70, Dox-CM: Doxorubicin-induced cardiomyopathy, dPWth: diastolic posterior wall thickness; E: E wave velocity; Ea: peak velocity of basal and lateral walls in early diastole;IVRT: isovolumic relaxation time; LV: left ventricle; LVEF: left ventricular ejection fraction; LVEV: left ventricular end volume; LVSF: left ventricularshortening fraction; Sa: peak velocity of basal and lateral walls in systole.doi:10.1371/journal.pone.0064711.g003
binding was blocked by incubating membranes in 5% non-fat dry
period, specific b1-, b2- or b3-AR functions and AC stimulation
milk in Tris-buffered saline (TBS) (200 mM Trizma base, 1.4 M
were assessed by constructing concentration-response curves to
NaCl, pH = 7.5) with 0.1% Tween 20 added (TBS-T) and then
different pharmacological agents. Thus, b1- and b2-AR functions
membranes were incubated with the primary antibody. Mem-
were assessed by perfusing isoproterenol (a non selective b-AR
branes were washed in 5% non-fat dry milk in TBS-T and
agonist, 1029 to 1025 M) in the presence of 1026 M L-748,337 (a
hybridized with the secondary antibody in 1% non-fat dry milk in
selective b3-AR antagonist) associated to 1026 M ICI-118,551 (a
TBS-T. Finally, membranes were washed with 5% non-fat dry
selective b2-AR antagonist) or to 1026 M CGP-20712A (a
milk in TBS-T then TBS, and antibody complexes were revealed
selective b1-AR antagonist), respectively. b3-AR function was
by the enhanced chemiluminescence detection process (Bio-Rad).
assessed by perfusing growing concentrations (1029 to 1025 M) of
Chemiluminescence was visualized using an Amersham Image-
SR 58611A (a b3-AR agonist) and AC response was assessed by
Quant RT-ECL camera (GE Healthcare) and band signals were
perfusing growing concentrations (3.1028 to 1025 M) of forskolin
assessed by densitometry with ImageQuant TL software (GE
(an AC activator). In order to assess the role of Gi in the b2-AR
Healthcare). For each lane, a ratio to the corresponding GAPDH
pathway, we used pertussis toxin (PTX) (a selective Gi inhibitor).
band intensity was calculated; the use of GAPDH as reference was
Classically, PTX is administered to rats two or three days before
validated by checking the abundance stability of GAPDH protein
experiments [22]. However, due to high mortality in Dox-CM rats
between Ctrl and Dox-CM groups.
pre-treated with PTX, we were not able to apply this protocol and
Antibody references and conditions used in this study are
used another one consisting in pre-treating heart with PTX
summarized in Table 1.
(4 mg.L21) in a closed system during 30 min, as described by othergroups [23]. Data were analysed using Datanalyst software
Whole Heart Contractility
(EMKA Technologies). After Langendorff experiments, hearts
At d35, after removal, hearts were quickly flushed into a cold
were weighed, as well as LV free walls which were carefully
Tyrode buffer, and then were mounted on a cannula via the aorta
separated from the heart.
on a Langendorff apparatus (EMKA Technologies) and perfusedwith a Krebs-modified solution (in mM: NaCl, 116; KCl, 5;
Data Analysis and Statistics
Data are presented as means 6 standard error to the mean
1.1; NaH2PO4, 0.35; NaHCO3, 27; glucose, 10;
mannitol, 16; Na-Pyruvate, 2; CaCl
(sem) of n experiments obtained from n different animals. In vivo
2, 1.8) continuously oxygen-
ated with a 95% O
and ex vivo cardiac parameters in baseline as well as protein
2, 5% CO2 gas mixture to maintain a pH 7.4.
Hearts were perfused at a constant flow-rate of 14 mL.min21 at
expressions were compared using Student's t test for unpaired
37.060.4uC. After heart beatings became regular, left auricle was
data. Concentration-response curves were compared with a two-
gently cut off to insert a small latex balloon, connected to a
way ANOVA (concentration, treatments) for repeated measures
pressure transducer to record LV pressure, into the LV via mitral
followed, when appropriated, by a Bonferroni's multiple compar-
valves. Before starting record, the minimum pressure into the
isons test. Differences were considered significant if P,0.05.
balloon was adjusted to 8–15 mmHg. Cardiac parameters wererecorded using IOX1.5.7 software (EMKA Technologies): inotro-
Drugs and Chemicals
pism, lusitropism and chronotropism were respectively evaluated
by measuring the contraction velocity (dP/dt
max), the relaxation
min) and the heart rate (HR). After the stabilization
dro chloride, L-748,337, N-[[3-[(2S)-2-Hydroxy-3-[[2-[4-[(phenylsulfo-
Table 3. In vivo cardiac parameters obtained by LV
catheterization in Ctrl and Dox-CM rats, at d35.
forskolin and PTX were obtained from Tocris Bioscience (Bristol,UK), isoproterenol was obtained from Sigma-Aldrich (St QuentinFallavier, France) and SR 58611A, [(RS)-N-[(25)-7-ethxycarbonyl-
ethanamide hydrochloride] was a generous gift from Sanofi-
Synthe´labo (Montpellier, France). All drugs were dissolved in distilled
water, excepted isoproterenol which was solubilised in 1% acid
ascorbic and L-748,337 and forskolin which were solubilised in
LV diastolic function
dimethylsulfoxide (Sigma-Aldrich). The final concentration of the
solvent in the organ bath was less than 0.1% v.v21 and was used as
controls for the effect of the active drug.
LV loading conditions LVEDP (mmHg)
Ctrl: control; Dox-CM: Doxorubicin-induced cardiomyopathy; dP/dtmax: leftventricular contraction velocity; dP/dtmin: left ventricular relaxation velocity; LV:
Mortality and General Characteristics of Animals
left ventricle; LVEDP: left ventricular end diastolic pressure; Tau: index of leftventricular relaxation constant. Values are means 6 sem. *: P,0.05 vs Ctrl.
Dox-CM rats did not gain weight during the Dox-treatment,
whereas the body weight of Ctrl rats increased regularly. After the
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Beta-Adrenoceptors in Doxorubicin Cardiomyopathy
Figure 4. b3-AR expression and function in Dox-CM hearts. A. Representative b3-AR immunoblotting at days 35 (d35) and 70 (d70). B. b3-ARprotein quantification at day 35. C. b3-AR protein quantification at day 70. Protein levels were quantified using Amersham ImageQuant RT-ECLcamera (GE Healthcare). The band signals were assessed by densitometry with ImageQuant TL software (GE Healthcare) and a ratio to thecorresponding GAPDH band intensity was calculated. D. Cardiac inotropic (dP/dtmax), E. lusitropic (dP/dtmin) and F. chronotropic (HR) effects ofincreasing concentrations of SR 58611A (1029 to 1025 M) were evaluated on isolated perfused hearts. **: P,0.001 vs Ctrl. Ctrl: control, Dox-CM:Doxorubicin-induced cardiomyopathy.doi:10.1371/journal.pone.0064711.g004
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Beta-Adrenoceptors in Doxorubicin Cardiomyopathy
end of Dox-treatment, the rat body weight increased again, but
intensity in Dox-CM and Ctrl (P = 0.192), whereas, at d70, b3-ARs
remained significantly lower than those of Ctrl rats, and reached a
were down-expressed in Dox-CM hearts compared to Ctrl hearts
plateau from 5 weeks after the end of Dox-treatment (day 49;
(237.267.9%; P,0.001) (Figures 4A–C). In Ctrl isolated heart, at
Figure 1A). In addition, Dox-CM rats became lethargic compared
d35, b3-AR stimulation performed by cumulative increasing
to Ctrl one.
concentrations of SR 58611A produced no significant effect on
The mortality rate was 25.0% at d35 and 58.3% at d70 in Dox-
heart rate and dP/dtmax at any concentration (P = 0.239 and
CM when no mortality was observed in Ctrl (Figure 1B). At d35,
P = 0.385, respectively) (Figures 4D, F) and decreased dP/dtmin
all surviving Dox-CM rats presented a hepatomegaly and 79.4%
only at the higher concentration of SR 58611A (1025M)
of them presented ascites (30.865.8 mL), whereas at d70 no
(P = 0.004) (Figure 4E). Compared to Ctrl hearts, b3-AR
hepatomegaly was observed and ascites was present in 38.9% of
stimulation in Dox-CM hearts produced a significant decrease in
Dox-CM rats (35.764.9 mL). Whole heart and LV weights from
dP/dtmax only at the higher concentration of SR 58611A (1025M)
Dox-CM rats were significantly smaller than those of Ctrl animals
(P,0.001) (Figure 4D) and had no effect on dP/dtmin nor heart
both at d35 and d70, and they were still smaller after
rate (P = 0.406 and P = 0.107, respectively) (Figures 4E, F).
normalization to the tibia length. However, heart/body weightratios and LV/body weight ratios were similar between Ctrl and
b1-adrenoceptor Expression and Function
Dox-CM rats at d35 when it was increased in Dox-CM group
By western-blot, the antibody directed against b1-AR detected a
compared to Ctrl at d70 (Table 2).
band at 72 kDa (Figure 5A) whose intensity was significantlydecreased in Dox-CM LV at d35 by 268.266.1% (P = 0.002 vs
Cardiac Function Obtained by Echocardiography-
Ctrl) (Figure 5B) and at d70 by 275.3614.0% (P,0.001 vs Ctrl)
(Figure 5C). In Ctrl heart, at d35, b1-AR stimulation induced a
Echocardiography-Doppler analyses were performed at d35
concentration-dependant increase in dP/dtmax, dP/dtmin and
and d70. Heart rate, measured before all echocardiographic
heart rate with a maximal effect observed at a concentration of
acquisitions, was monitored in order to be similar between both
1025 M (P,0.001, each) (Figures 5D–F). The heart rate and the
groups (d35: Ctrl: 36466 bpm, Dox-CM: 37166 bpm, P = 0.427;
positive inotropic and lusitropic effects were unchanged in Dox-
d70: Ctrl: 36866 bpm, Dox-CM: 337617 bpm, P = 0.079).
CM isolated hearts compared to Ctrl hearts (P = 0.544, P = 0.772
Representative images obtained by echocardiography in short
and P = 0.667, respectively) (Figures 5D–F).
axis view of a two-dimensionally directed M-mode at d35 areshown in Figure 2.
b2-adrenoceptor Expression and Function
The dPWth decreased in Dox-CM compared to Ctrl hearts at
By western-blot, the antibody directed against b2-AR detected a
d35 (29.563.3%, P = 0.055) and was significantly thinner at d70
band at 55 kDa (Figure 6A) whose intensity was significantly
(220.564.4%, P,0.001) (Figure 3A). LVESV was similar in both
increased in Dox-CM LV at d35 by +57.9621.6% (P = 0.039 vs
groups at d35 as at d70 (P = 0.199 and P = 0.116, respectively). On
Ctrl) (Figure 6B) and at d70 by +173.7635.1% (P,0.001 vs Ctrl)
the contrary, LVEDV was reduced in Dox-CM compared to Ctrl
(Figure 6C). At d35, b2-AR stimulation induced a similar
at d35 (212.661.9%, P,0.001) as at d70 (221.768.7%,
concentration-dependant increase of heart rate between Ctrl and
P = 0.050) (Figure 3B).
Dox-CM hearts (P = 0.695) (Figure 6F). In Ctrl isolated hearts, b2-
Compared to Ctrl, Dox-CM LVs were characterized at d35 by
AR stimulation induced a concentration-dependant increase of
mild but significant decreases in LVEF (-14.262.0%; P,0.001)
dP/dtmax and dP/dtmin (P,0.001 and P = 0.005, respectively)
and LVSF (223.462.7%; P,0.001), that were more pronounced
(Figures 6D, E) but at a lesser extent than b1-AR one. In Dox-CM
at d70 (225.266.2%, P,0.001 and 234.967.4%, P,0.001,
hearts, the maximal effects induced by b2-AR stimulation on dP/
respectively) (Figures 3C, D). Moreover, Dox-CM hearts presented
dtmax and dP/dtmin, compared to Ctrl, were both increased by
an alteration in longitudinal deformation, since Sa was decreased
+108.0618.2% (P,0.001) and +155.3626.8% (P,0.001), respec-
at d35 (231.266.5%; P = 0.001) as at d70 (248.6610.3%,
tively (Figures 6D, E), and reached a similar level than this
P,0.001) (Figure 3E) whereas radial deformation was only
produced by b1-AR stimulation.
observed at d70 (238.764.9%, P = 0.031) (Figure 3F). In Dox-CM rats, IVRT value was significantly increased at d35
Gs Protein Expression and Adenylyl Cyclase Stimulation
(+25.763.4%, P,0.001) as at d70 (+31.1610.6%, P = 0.003)
By western-blot, we detected two bands for Gsa protein
(Figure 3G) albeit the LV filling pressures, evaluated by E/Ea
expression: a short one at 45 kDa and a long one at 52 kDa
ratio, were similar in Ctrl and Dox-CM rats at both d35 and d70
(Figure 7A). The expression of both forms was unchanged in Dox-
(P = 0.776 and P = 0.071, respectively) (Figure 3H).
CM hearts at both d35 and d70 (Figures 7B, C). In Ctrl and Dox-CM isolated hearts, at d35, forskolin induced a similar concen-
Cardiac Function Obtained by Left Ventricle
tration-dependant increase of heart rate between Ctrl and Dox-
CM hearts (P = 0.941) (Figure 7F). In Ctrl group, forskolin induced
As our study consists to identify molecular target at early stage
a concentration-dependant increase of dP/dtmax (P,0.001) and
of Dox-CM, we only performed LV catheterization at d35. Basal
dP/dtmin (P = 0.010) (Figures 7D, E). In Dox-CM isolated hearts
heart rate, measured before all pressure acquisitions, was increased
compared to Ctrls, the forskolin effects on dP/dtmax and dP/dtmin
in Dox-CM rats compared to Ctrl (P = 0.007) (Table 3). Dox-CM
were increased by +43.2610.3% (P = 0.004) and +61.1610.7%
rats presented a decreased Tau (210.062.2%; P = 0.009) albeit
(P = 0.002), respectively (Figures 7D, E).
dP/dtmax and dP/dtmin were unchanged (Table 3). LVEDP wasunchanged between both groups (P = 0.216).
Gi Protein Expression and its Involvement in b2-ARCardiac Contractility
b3-adrenoceptor Expression and Function
Gia2 protein expression was detected by one band of 35 kDa
Using western-blot, we detected b3-AR protein at a band of
(Figure 8A) whose intensity was similar both in Ctrl and Dox-CM
68 kDa (Figure 4A). At d35, the detected band presented a similar
hearts at d35 (P = 0.184 vs Ctrl) (Figure 8B), but was increased in
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May 2013 Volume 8 Issue 5 e64711
Beta-Adrenoceptors in Doxorubicin Cardiomyopathy
Figure 5. Impaired b1-AR expression in Dox-CM hearts was not associated to an impaired cardiac b1-AR function. A. Representative b1-AR immunoblotting at days 35 (d35) and 70 (d70). B. b1-AR protein quantification at day 35. C. b1-AR protein quantification at day 70. Protein levelswere quantified using Amersham ImageQuant RT-ECL camera (GE Healthcare). The band signals were assessed by densitometry with ImageQuant TLsoftware (GE Healthcare) and a ratio to the corresponding GAPDH band intensity was calculated. D. Cardiac inotropic (dP/dtmax), E. lusitropic (dP/dtmin) and F. chronotropic (HR) effects of increasing concentrations of isoproterenol (1029 to 1025 M) in the presence of 1026 M ICI-118,551 a b2-ARantagonist and 1026 M of L-748,337 a b3-AR antagonist were evaluated on isolated perfused hearts. B: Baseline; B+A; Baseline in presence ofantagonists. **: P,0.001 vs Ctrl. Ctrl: control, Dox-CM: Doxorubicin-induced cardiomyopathy.doi:10.1371/journal.pone.0064711.g005
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May 2013 Volume 8 Issue 5 e64711

Target cell availability and the successful suppression of HIV by hydroxyurea and didanosine Rob J. De Boer AIDS 1998, 12:1567–1570 Keywords: Hydroxyurea, immunosuppression, target cell availability, 72 weeks of ddI–HU treatment, three out of sixpatients had no detectable plasma virus, and that there